Whooping Cough

Bordetella pertussis uniquely colonizes the cilia of the mammalian respiratory epithelium (Figures 1 and 2); it does not invade tissues. The bacterium is a pathogen for humans and possibly for higher primates, and no other reservoir is known. Whooping cough is a relatively mild disease in adults but has a significant mortality rate in infants. Until immunization was introduced in the 1930s, whooping cough was one of the most frequent and severe diseases of infants in the United States.

Pathogenesis

The disease pertussis has two stages. The first stage, colonization, is an upper respiratory disease with fever, malaise and coughing, which increases in intensity over about a 10-day period. During this stage the organism can be recovered in large numbers from pharyngeal cultures, and the severity and duration of the disease can be reduced by antimicrobial treatment. Adherence mechanisms of B. pertussis involve a "filamentous hemagglutinin" (FHA), which is a fimbrial-like structure on the bacterial surface, and cell-bound pertussis toxin (PT). Short range effects of soluble toxins play a role as well in invasion during the colonization stage.

Figure 1. Colonization of tracheal epithelial cells by Bordetella pertussis

The second or toxemic stage of pertussis follows relatively nonspecific symptoms of the colonizaton stage. It begins gradually with prolonged and paroxysmal coughing that often ends in a characteristic inspiratory gasp (whoop). During the second stage, B. pertussis can rarely be recovered, and antimicrobial agents have no effect on the progress of the disease. This stage is mediated by a variety of soluble toxins.

Colonization

Studies of B. pertussis and its adhesins have focused on cultured mammalian cells that lack most of the features of ciliated epithelial cells. However, some generalities have been drawn. The two most important colonization factors are the filamentous hemagglutinin (FHA) and the pertussis toxin (PT). Filamentous hemagglutinin is a large (220 kDa) protein that forms filamentous structures on the cell surface. FHA binds to galactose residues on a sulfated glycolipid called sulfatide which is very common on the surface of ciliated cells. Mutations in the FHA structural gene reduce the ability of the organism to colonize, and antibodies against FHA provide protection against infection. However, it is unlikely that FHA is the only adhesin involved in colonization. The structural gene for FHA has been cloned and expressed in E. coli, raising the possibility of its production for use in a component vaccine.

One of the toxins of B. pertussis, the pertussis toxin (PT), is also involved in adherence to the tracheal epithelium. Pertussis toxin is a 105 kDa protein composed of six subunits: S1, S2, S3, (2)S4, and S5 (Figure 3). The toxin is both secreted into the extracellular fluid and cell bound. Some components of the cell-bound toxin (S2 and S3) function as adhesins, and appear to bind the bacteria to host cells. S2 and S3 utilize different receptors on host cells. S2 binds specifically to a glycolipid called lactosylceramide, which is found primarily on the ciliated epithelial cells. S3 binds to a glycoprotein found mainly on phagocytic cells.

The S1 subunit of pertussis toxin is the A component with ADP ribosylating activity, and the function of S2 and S3 is presumed to be involved in binding the intact (extracellular) toxin to its target cell surface. Antibodies against PT components prevent colonization of ciliated cells by the bacteria and provide effective protection against infection. Thus, pertussis toxin is clearly an important virulence factor in the initial colonization stage of the infection.

Since the S3 subunit of pertussis toxin is able to bind to the surface of phagocytes, and since FHA will attach to integrin CR3 on phagocyte surfaces (the receptor for complement C3b), it has been speculated that the bacterium might bind preferentially to phagocytes in order to facilitate its own engulfment. The role of such self-initiated phagocytosis is not clear. Bacteria taken up by this abnormal route may avoid stimulating the oxidative burst that normally accompanies phagocytic uptake of bacterial cells which are opsonized by antibodies or complement C3b. Once inside of cells the bacteria might utilize other toxins (i.e. adenylate cyclase toxin) to compromise the bactericidal activities of phagocytes. In any case, there is some evidence that Bordetella pertussis can use this mechanism to get into and to persist in phagocytes as an intracellular parasite. If B. pertussis is an intracellular parasite it would explain why immunity to pertussis correlates better with the presence of specific cytotoxic T cells than it does with the presence of antibodies to bacterial products.

B. pertussis produces at least two other types of adhesins, two types of fimbriae and a nonfimbrial surface protein called pertactin, but their role in adherence and pathogenesis is not well established.

Toxins Produced by B. pertussis

B. pertussis produces a variety of substances with toxic activity in the class of exotoxins and endotoxins. (see Figure 6).

1. It secretes its own invasive adenylate cyclase which enters mammalian cells (Bacillus anthracis produces a similar enzyme, EF). This toxin acts locally to reduce phagocytic activity and probably helps the organism initiate infection. This toxin is a 45 kDa protein that may be cell-associated or released into the environment. Mutants of B. pertussis in the adenylate cyclase gene have reduced virulence in mouse models. The organisms can still colonize but cannot produce the lethal disease. The adenylate cyclase toxin is a single polypeptide with an enzymatic domain (i.e., adenylate cyclase activity) and a binding domain that will attach to host cell surfaces. The adenylate cyclase was originally identified as a hemolysin because it will lyse red blood cells. In fact, it is responsible for hemolytic zones around colonies of Bordetella pertussis growing on blood agar. Probably it inserts into the erythrocyte membrane which causes hemolysis. An interesting feature of the adenylate cyclase toxin is that it is active only in the presence of a eukaryotic regulatory molecule called calmodulin, which up-regulates the activity of the eukaryotic adenylate cyclase. The adenylate cyclase toxin is only active in the eukaryotic cell since no similar regulatory molecule exists in procaryotes. Thus, the molecule seems to have evolved specifically to parasitize eukaryotic cells. Anthrax EF (edema factor) is also a calmodulin-dependent adenylate cyclase.

    

2. It produces a highly lethal toxin (formerly called dermonecrotic toxin) which causes inflammation and local necrosis adjacent to sites where B. pertussis is located. The lethal toxin is a 102 kDa protein composed of four subunits, two with a mw of 24kDa and two with mw of 30 kDa. It causes necrotic skin lesions when low doses are injected subcutaneosly in mice and is lethal in high doses. The role of the toxin in whooping cough is not known.

    

3. It produces a substance called the tracheal cytotoxin which is toxic for ciliated respiratory epithelium and which will stop the ciliated cells from beating. This substance is not a classic bacterial exotoxin since it is not composed of protein. The tracheal cytotoxin is a peptidoglycan fragment, which appears in the extracellular fluid where the bacteria are actively growing. The toxin kills ciliated cells and causes their extrusion from the mucosa. It also stimulates release of cytokine IL-1, and so causes fever.

    

4. It produces the pertussis toxin, PT, a protein that mediates both the colonization and toxemic stages of the disease. PT is a two component, A+B bacterial exotoxin. The A subunit (S1) is an ADP ribosyl transferase. The B component, composed of five polypeptide subunits (S2 through S5), binds to specific carbohydrates on cell surfaces (Figure 3). The role of PT in invasion has already been discussed. PT is transported from the site of growth of the Bordetella to various susceptible cells and tissues of the host. Following binding of the B component to host cells, the A subunit is inserted through the membrane and released into the cytoplasm in a mechanism of direct entry (Figure 5). The A subunit gains enzymatic activity and transfers the ADP ribosyl moiety of NAD to the membrane-bound regulatory protein Gi that normally inhibits the eukaryotic adenylate cyclase. The Gi protein is inactivated and cannot perform its normal function to inhibit adenylate cyclase. The conversion of ATP to cyclic AMP cannot be stopped and intracellular levels of cAMP increase (Figure 4). This has the effect to disrupt cellular function, and in the case of phagocytes, to decrease their phagocytic activities such as chemotaxis, engulfment, the oxidative burst, and bacteridcidal killing. Systemic effects of the toxin include lymphocytosis and alteration of hormonal activities that are regulated by cAMP, such as increased insulin production (resulting in hypoglycemia) and increased sensitivity to histamine (resulting in increased capillary permeability, hypotension and shock). PT also affects the immune system in experimental animals. B cells and T cells that leave the lymphatics show an inability to return. This alters both AMI and CMI responses and may explain the high freqency of secondary infections that accompany pertussis (the most frequent secondary infections during whooping cough are pneumomia and otitis media).

   Although the effects of the pertussis toxin are dependent on ADP ribosylation, it has been shown that mere binding of the B oligomer can elicit a response on the cell surface such as lymphocyte mitogenicity, platelet activation, and production of insulin effects.

   The pertussis toxin gene has been cloned and sequenced and the subunits expressed in E. coli. The toxin can be inactivated and converted to toxoid for use in component vaccines.

   Figure 2. Comparison between cholera toxin and pertussis toxin (ptx) in their ability to interfere with the regulation of the eukaryotic adenylate cyclase complex.

   Normal regulation of adenylate cyclase activity in mammalian cells (Adenylate cyclase (AC) is activated normally by a stimulatory regulatory protein (Gs) and guanosine triphosphate (GTP); however the activation is normally brief because an inhibitory regulatory protein (Gi) hydrolyzes the GTP.

   Adenylate cyclase activated by cholera toxin (The cholera toxin A1 fragment catalyzes the attachment of ADP-Ribose (ADPR) to the regulatory protein Gs, forming Gs-ADPR from which GTP cannot be hydrolyzed. Since GTP hydrolysis is the event that inactivates adenylate cyclase (AC), the enzyme remains continually activated.

   

   Adenylate cyclase activated by pertussis toxin (The pertussis A subunit transfers the ADP ribosyl moiety of NAD to the membrane-bound regulatory protein Gi that normally inhibits the eukaryotic adenylate cyclase. The Gi protein is inactivated and cannot perform its normal function to inhibit adenylate cyclase. The conversion of ATP to cyclic AMP cannot be stopped.)

   

5. Lipopolysaccharide. As a Gram-negative bacterium Bordetella pertussis possesses lipopolysaccharide (endotoxin) in its outer membrane, but its LPS is unusual. It is heterogeneous, with two major forms differing in the phosphate content of the lipid moiety. The alternative form of Lipid A is designated Lipid X. The unfractionated material elicits the usual effects of LPS (i.e., induction of IL-1, activation of complement, fever, hypotension, etc.), but the distribution of those activities is different in the two forms of LPS. For example, Lipid X, but not Lipid A, is pyrogenic, and its O-side chain is a very powerful immune adjuvant. Furthermore, Bordetella LPS is more potent in the limulus assay than LPS from other Gram-negative bacteria, so it is not reliable to apply knowledge of the biological activity of LPS in the Enterobacteriaceae to the LPS of Bordetella. The role of this unusual LPS in the pathogenesis of whooping cough has not been investigated.

Regulation of Virulence Factors in B. pertussis

The production of virulence factors in B. pertussis is regulated in several different ways. Expression of virulence factors is regulated by the bvg operon.

First, the organisms undergo an event called phase variation resulting in the loss of most virulence factors and some undefined outer membrane proteins. Phase variation has been shown to occur at a genetic frequency of 10-4 - 10-6 generations and results from a specific DNA frame shift that comes about after the insertion of a single nucleotide into the bvg (also known as vir) operon.

A similar process called phenotypic modulation, occurs in response to environmental signals such as temperature or chemical content, and is reversible. This is an adaptive process mediated by the products of the bvg operon, and is an example of a two-component environmental-sensing (regulatory) system used by other bacteria. The expression of these regulatory proteins is itself regulated by environmental signals, such that entry into a host might induce components required for survival and production of disease.

The Whooping Cough Vaccine

The development of the whooping cough vaccine in the 1950s has made whooping cough an uncommon disease in developed countries. In countries where the vaccine is not used whooping cough is an important cause of mortality in children, with an estimated 51,000,000 cases and 600,000 deaths annually.

Historically, the whooping cough vaccine has been administered as a merthiolate-killed bacterial cell suspension which is part of the DTP vaccine (The P in DTP stands for Pertussis cells). Unfortunately, about 20% of the children that receive the whole cell vaccine experience mild side effects. About 0.1% of infants experience convulsions soon after receiving the vaccine and in a very small number of cases (1 in 150,000?) severe or irreversible brain damage may occur. In the absence of the disease in an immune population, parents have begun to wonder if the risk of vaccinating children outweighs the risk of the disease, and the value of the whole cell vaccine has been questioned.

Several new acellular vaccines have been developed from purified components of B. pertussis. Demonstration of the protective effects of anti-PT and anti-FHA antibodies in the mouse model, focused vaccine production on combinations of inactivated pertussis toxin (toxoid) and filamentous hemagglutinin. Multicomponent acellular vaccines containing combinations of pertussis toxoid, filamentous hemagglutinin, pertactin, and the two types of fimbriae, are now being used in several countries including the U.S. The new vaccine, known as acellular pertussis has fewer side effects than the whole cell vaccine and is currently recommended for use under the conditions described below.

For decades, the pertussis vaccine has been given in combination with vaccines against diphtheria and tetanus. The combination is known as the DTP vaccine. Recently, infants have been able to receive a vaccine that combines the DTP vaccine with the vaccine against Haemophilus influenzae type b meningitis (Hib). This vaccine is called DTPH. The diphtheria-tetanus-pertussis vaccine using acellular pertussis is known as DTaP. The diphtheria-tetanus-pertussis vaccination is given in five doses: at 2, 4, 6, 12-18 months and 4-6 years of age. Previously, DTaP had been recommended only for the fourth and fifth doses. Following FDA licensure of DTaP for infants, the Advisory Committee on Immunization Practices of the United States Public Health Service now recommends that DTaP be used for the first four doses and that DTaP still be used for the fourth and fifth doses for children who received DTP in their first three doses. The Committee is awaiting study results before making a recommendation for the fifth dose for children who now will receive DTaP in their first four doses. The recommendation still permits the use of DTP and DTPH--the combination that includes the vaccine against Haemophilus influenzae type b meningitis.